Methods of processing lignocellulosic biomass using single-stage autohydrolysis and enzymatic hydrolysis with c5 bypass and post-hydrolysis

ABSTRACT

The invention relates, in general, to methods of processing Lignocellulosic biomass to fermentable sugars and to methods that rely on hydrothermal pretreatment. Xylose monomer yields comparable to those achieved using two-stage pretreatments can be achieved from soft Lignocellulosic biomass feedstocks by pretreasting to very low severity in a single-stage pressurized hydrothermal pretreatment, followed by enzymatic hydrolysis to release xylose retained in the solid state. In some embodiments, pretreated biomass is separated into a solid fraction and a liquid fraction, the solid fraction subjected to enzymatic hydrolysis, and the separated liquid fraction subsequently mixed with the hydrolysed solid fraction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/418,667, filed Jan. 30, 2015, which is a national stage filing under35 U.S.C. 371 of PCT/DK2013/050256, filed Aug. 1, 2013, whichInternational Application was published by the International Bureau inEnglish on Feb. 6, 2014, and application claims priority from U.S.Provisional Application No. 61/678,130 filed Aug. 1, 2012, and DenmarkApplication No. PA 2012 70461, filed Aug. 1, 2012, which applicationsare hereby incorporated in their entirety by referenced in thisapplication.

FIELD

The invention relates, in general, to methods of processinglignocellulosic biomass to fermentable sugars and, in particular, tomethods that rely on hydrothermal pretreatment.

BACKGROUND

Historical reliance on petroleum and other fossil fuels has beenassociated with dramatic and alarming increases in atmospheric levels ofgreenhouse gases. International efforts are underway to mitigategreenhouse gas accumulation, supported by formal policy directives inmany countries. One central focus of these mitigation efforts has beendevelopment of processes and technologies for utilization of renewableplant biomass to replace petroleum as a source of precursors for fuelsand other chemical products. The annual growth of plant-derived biomasson earth is estimated to approximate 1×10{circle around ( )}11 metrictons per year dry weight. See Lieth and Whittaker (1975). Biomassutilization is, thus, an ultimate goal in development of sustainableeconomy.

Fuel ethanol produced from sugar and starch based plant materials, suchas sugarcane, root and grain crops, is already in wide use, with globalproduction currently topping 73 billion liters per year. Ethanol hasalways been considered an acceptable alternative to fossil fuels, beingreadily usable as an additive in fuel blends or even directly as fuelfor personal automobiles. However, use of ethanol produced by these“first generation” bioethanol technologies does not actually achievedramatic reduction in greenhouse gas emission. The net savings is onlyabout 13% compared with petroleum, when the total fossil fuel inputs tothe final ethanol outputs are all accounted. See Farrell et al. (2006).

Moreover, both economic and moral objections have been raised to the“first generation” bioethanol market. This effectively places demand forcrops as human food into direct competition with demand for personalautomobiles. And indeed, fuel ethanol demand has been associated withincreased grain prices that have proved troublesome for poor,grain-importing countries.

Great interest has arisen in developing biomass conversion systems thatdo not consume food crops—so-called “second generation” biorefining,whereby bioethanol and other products can be produced fromlignocellulosic biomass such as crop wastes (stalks, cobs, pits, stems,shells, husks, etc . . . ), grasses, straws, wood chips, waste paper andthe like.

In “second generation” technology, fermentable 6-carbon (C6) sugarsderived primarily from cellulose and fermentable 5-carbon (C5) sugarsderived from hemicellulose are liberated from biomass polysaccharidepolymer chains by enzymatic hydrolysis or, in some cases, by purechemical hydrolysis. The fermentable sugars obtained from biomassconversion in a “second generation” biorefinery can be used to producefuel ethanol or, alternatively, other fuels such as butanol, or lacticacid monomers for use in synthesis of bioplastics, or many otherproducts.

The total yield of both C5 and C6 sugars is a central consideration incommercialization of lignocellulosic biomass processing. In the case ofethanol production, and also production of lactate or other chemicals,it can be advantageous to combine both C5 and C6 sugar process streamsinto one sugar solution. Modified fermentive organisms are now availablewhich can efficiently consume both C5 and C6 sugars in ethanolproduction. See e.g. Madhavan et al. (2012); Dumon et al. (2012); Hu etal. (2011); Kuhad et al. (2011); Ghosh et al. (2011); Kurian et al.(2010); Jojima et al. (2010); Sanchez et al. (2010); Bettiga et al.(2009); Matsushika et al. (2009).

Because of limitations of its physical structure, lignocellulosicbiomass cannot be effectively converted to fermentable sugars byenzymatic hydrolysis without some pretreatment process. A wide varietyof different pretreatment schemes have been reported, each offeringdifferent advantages and disadvantages. For review see Agbor et al.(2011); Girio et al. (2010); Alvira et al. (2010); Taherzadeh and Karimi(2008). From an environmental and “renewability” perspective,hydrothermal pretreatments are especially attractive. These utilizepressurized steam/liquid hot water at temperatures on the order of160-230° C. to gently melt hydrophobic lignin that is intricatelyassociated with cellulose strands, to solubilize a major component ofhemicellulose, rich in C5 sugars, and to disrupt cellulose strands so asto improve accessibility to productive enzyme binding.

Hydrothermal pretreatments can be conveniently integrated with existingcoal- and biomass-fired electrical power generation plants toefficiently utilize turbine steam and “excess” power productioncapacity.

In the case of hydrothermal processes, it is well known in the art, andhas been widely discussed, that pretreatment must be optimized betweenconflicting purposes. On the one hand, pretreatment should ideallypreserve hemicellulose sugar content, so as to maximize the ultimateyield of monomeric hemicellulose-derived sugars. Yet at the same time,pretreatment should sufficiently expose and pre-condition cellulosechains to susceptibility of enzymatic hydrolysis such that reasonableyields of monomeric cellulose-derived sugars can be obtained withminimal enzyme consumption. Enzyme consumption is also a centralconsideration in commercialization of biomass processing, which teeterson the verge of “economic profitability” in the context of “globalmarket economies” as these are currently defined. Notwithstandingdramatic improvements in recent years, the high cost of commerciallyavailable enzyme preparations remains one of the highest operating costsin biomass conversion.

As hydrothermal pretreatment temperatures and reactor residence timesare increased, a greater proportion of C5 sugars derived from hemicellulose is irretrievably lost due to chemical transformation to othersubstances, including furfural and products of condensation reactions.Yet higher temperatures and residence times are required in order toproperly condition cellulose fibers for efficient enzymatic hydrolysisto monomeric 6-carbon glucose.

In the prior art, an often used parameter of hydrothermal pretreatment“severity” is “R₀,” which is typically referred to as a log value. Roreflects a composite measure of pretreatment temperature and reactorresidence time according to the equation: R₀=t*EXP[(T-100)/14.75] wheret is residence time in minutes and T is reaction temperature in degreescentigrade. We have developed an alternative measure of pretreatmentseverity, “xylan number,” which provides a negative linear correlationwith classical log Ro, even at very low levels of “severity.” Unlike Ro,which is a purely empirical description of pretreatment conditions,xylan number is a functionally significant physical parameter. Xylannumber provides a measure of pretreatment degree that permits comparisonof divergent biomass feedstocks, in terms of C5 recoveries, regardlessof the Ro severity to which they have been subjected.

Whether hydrothermal pretreatment severity is expressed in terms of“xylan number” or “R₀,” the optimization of pretreatment conditions forany given biomass feedstock inherently requires some compromise betweendemands for high monomeric C5 sugar yields from hemicellulose (lowseverity) and the demands for high monomeric C6 sugar yields fromcellulose (high severity).

Hem icellulose-derived C5 sugars solubilized during hydrothermalpretreatment typically include a large fraction of xylo-oligomers, whichstrongly inhibit cellulase enzyme catalysis. See Shi et al. (2011);Quing and Wyman (2011); Quing et al. (2010). Other soluble byproducts ofpretreatment, including acetic acid and phenolic compounds derived fromsolubilized lignin, are also known to inhibit cellulase enzymecatalysis. See Kothari and Lee (2011); Ximenes et al. (2010). Thepresence of effective levels of enzyme inhibitors increases enzymeconsumption required to achieve a given degree of hydrolysis.Accordingly, “economic profitability” of commercial scale biomassconversion favors minimization of cellulase inhibition by solublecompounds derived from pretreatment.

A variety of different hydrothermal pretreatment strategies have beenreported for maximizing sugar yields from both hem icellulose andcellulose and for minimizing xylo-oligomer inhibition of cellulasecatalysis. In some cases, exogenous acids or bases are added in order tocatalyse hem icellulose degradation (acid; pH<3.5) or ligninsolubilisation (base; pH>9.0). In other cases, hydrothermal pretreatmentis conducted using only very mild acetic acid derived fromlignocellulose itself (pH 3.5-9.0). Hydrothermal pretreatments underthese mild pH conditions have been termed “autohydrolysis” processes,because acetic acid liberated from hem icellulose esters itself furthercatalyses hem icellulose hydrolysis.

Acid catalysed hydrothermal pretreatments, known as “dilute acid” or“acid impregnation” treatments, typically provide high yields of C5sugars, since comparable hem icellulose solubilisation can occur atlower temperatures in the presence of acid catalyst. Total C5 sugaryields after dilute acid pretreatment followed by enzymatic hydrolysisare typically on the order of 75% or more of what could theoretically beliberated from any given biomass feedstock. See e.g. Baboukaniu et al.(2012); Won et al. (2012); Lu et al. (2009); Jeong et al. (2010); Lee etal. (2008); Sassner et al. (2008); Thomsen et al. (2006); Chung et al.(2005).

Autohydrolysis hydrothermal pretreatments, in contrast, typicallyprovide much lower yields of C5 sugars, since higher temperaturepretreatment is required in the absence of acid catalyst. With theexception of autohydrolysis pretreatment conducted at commerciallyunrealistic low dry matter content, autohydrolysis treatments typicallyprovide C5 sugar yields <40% theoretical recovery. See e.g. Diaz et al.(2010); Dogaris et al. (2009). C5 yields from autohydrolysis as high as53% have been reported in cases where commercially unrealistic reactionstimes and extreme high enzyme doses were used. But even these very highC5 yields remain well beneath levels routinely obtained using diluteacid pretreatment. See e.g. Lee et al. (2009); Ohgren et al. (2007).

As a consequence of lower C5 yields obtained with autohydrolysis, mostreports concerning hydrothermal pretreatment in commercial biomassconversion systems have focused on dilute acid processes.Hemicellulose-derived C5 sugar yields on the order of 85% have beenachieved through use of so-called “two-stage” dilute acid pretreatments.In two-stage pretreatments, a lower initial temperature is used tosolubilize hem icellulose, whereafter the C5-rich liquid fraction isseparated. In the second stage, a higher temperature is used tocondition cellulose chains. See e.g. Mesa et al. (2011); Kim et al.(2011); Chen et al. (2010); Jin et al. (2010); Monavari et al. (2009);Soderstrom et al.

(2005); Soderstrom et al. (2004); Soderstrom et al. (2003); Kim et al.(2001); Lee et al. (1997); Paptheofanous et al. (1995). One elaborate“two-stage” dilute acid pretreatment system reported by the US NationalRenewable Energy Laboratory (NREL) claims to have achieved C5 yields onthe order of 90% using corn stover as feedstock. See Humbird et al.(2011).

Xylo-oligomer inhibition of cellulase catalysis is avoided in diluteacid systems because hydrolysis of xylo-oligomers to monomeric xylose iscatalysed by the added acid. The acid catalysed hydrolysis ofxylo-oligomers also occurs within a separate process stream from thatstream in which residual solids are subject to enzymatic hydrolysis.

Notwithstanding the lower C5 yields which it provides, autohydrolysiscontinues to offer competitive advantages over dilute acid pretreatmentson commercial scale.

Most notable amongst the advantages of autohydrolysis processes is thatthe residual, unhydrolysed lignin has greatly enhanced market valuecompared with lignin recovered from dilute acid processes. First, thesulphuric acid typically used in dilute acid pretreatment imparts aresidual sulphur content. This renders the resulting lignin unattractiveto commercial power plants which would otherwise be inclined to consumesulphur-free lignin fuel pellets obtained from autohydrolysis as a“green” alternative to coal.

Second, the sulfonation of lignin which occurs during sulphuricacid-catalysed hydrothermal pretreatments renders it comparativelyhydrophilic, thereby increasing its mechanical water holding capacity.This hydrophilicity both increases the cost of drying the lignin forcommercial use and also renders it poorly suited for outdoor storage,given its propensity to absorb moisture. So-called “techno-economicmodels” of NREL's process for lignocellulosic biomass conversion, withdilute acid pretreatment, do not even account for lignin as a saleablecommodity—only as an internal source of fuel for process steam. SeeHumbird et al. (2011). In contrast, the “economic profitability” ofprocess schemes that rely on autohydrolysis include a significantcontribution from robust sale of clean, dry lignin pellets. This isespecially significant in that typical soft lignocellulosic biomassfeedstocks comprise a large proportion of lignin, between 10 and 40% ofdry matter content. Thus, even where process sugar yields fromautohydrolysis systems can be diminished relative to dilute acidsystems, overall “profitability” can remain equivalent or even better.

Autohydrolysis processes also avoid other well known disadvantages ofdilute acid. The requirement for sulphuric acid diverges from aphilosophical orientation favouring “green” processing, introduces asubstantial operating cost for the acid as process input, and creates aneed for elaborate waste water treatment systems and also for expensiveanti-corrosive equipment.

Autohydrolysis is also advantageously scalable to modest processingscenarios. The dilute acid process described by NREL is so complex andelaborate that it cannot realistically be established on a smallerscale—only on a gigantic scale on the order of 100 tons of biomassfeedstock per hour. Such a scale is only appropriate inhyper-centralized biomass processing scenarios. See Humbird et al.(2011). Hyper-centralized biomass processing of corn stover may well beappropriate in the USA, which has an abundance of genetically-engineeredcorn grown in chemically-enhanced hyper-production. But such a system isless relevant elsewhere in the world. Such a system is inappropriate formodest biomass processing scenarios, for example, on-site processing atsugar cane or palm oil or sorghum fields, or regional processing ofwheat straw, which typically produces much less biomass per hectare thancorn, even with genetic-engineering and chemical-enhancements.

Autohydrolysis systems, in contrast with dilute acid, are legitimately“green,” readily scalable, and unencumbered by requirements forelaborate waste water treatment systems. It is accordingly advantageousto provide improved autohydrolysis systems, even where these may not beobviously advantageous over dilute acid systems in terms of sugar yieldsalone.

The problem of poor C5 monomer yields with autohydrolysis has generallydriven commercial providers of lignocellulosic biomass processingtechnology to pursue other approaches. Some “two-stage” pretreatmentsystems, designed to provide improved C5 yields, have been reported withautohydrolysis pretreatments. See WO2010/113129; US2010/0279361; WO2009/108773; US2009/0308383; U.S. Pat. No. 8,057,639; US20130029406.

In these “two stage” pretreatment schemes, some C5-rich liquid fractionis removed by solid/liquid separation after a lower temperaturepretreatment, followed by a subsequent, higher temperature pretreatmentof the solid fraction. Most of these published patent applications didnot report actual experimental results. In its description of two-stageautohydrolytic pretreatment in WO2010/113129, Chemtex Italia reports atotal of 26 experimental examples using wheat straw with an average C5sugar recovery of 52%. These C5 recovery values do not distinguishbetween C5 recovery per se and monomer sugar yields, which is thesubstrate actually consumed in fermentation to ethanol and other usefulproducts.

The introduction of a second pretreatment stage into a scheme forprocessing lignocellulosic biomass introduces additional complexitiesand costs. It is accordingly advantageous to substantially achieve theadvantages of two-stage pretreatment using a simple single-stageautohydrolysis system.

We have discovered that, where single-stage autohydrolysis pretreatmentis conducted to very low severity, unexpectedly high final C5 monomeryields of 60% theoretical yield and higher can be achieved followingenzymatic hydrolysis, while still achieving reasonable glucose yields.Where biomass feedstocks are pretreated to xylan number 10% and higher,a large amount of the original xylan content remains within the solidfraction. Contrary to expectations, this very high residual xylancontent can be enzymatically hydrolysed to monomer xylose, with highrecovery, while sacrificing only a very small percentage of celluloseconversion to glucose.

At these very low severity levels, the production of soluble by-productsthat affect cellulase activity or fermentive organisms is kept so lowthat the pretreated material can be used directly in enzymatichydrolysis, and subsequent fermentation, typically without requirementfor any washing or other de-toxification step.

Inhibition of cellulase catalysis by xylo-oligomers or by other solubleproducts in the liquid fraction can be easily avoided in the process. Asolid/liquid separation step following pretreatment generates a liquidfraction and a solid fraction. The C5-rich liquid fraction is maintainedseparately in “bypass” from the solid fraction during enzymatichydrolysis. Following enzymatic hydrolysis of the solid fraction, liquidfraction is added to hydrolysate and subject to post-hydrolysis byremaining active xylanase enzymes. Xylo-oligomers within the liquidfraction are in this manner hydrolysed to xylose monomers only aftercellulase activity is no longer necessary. The resulting combinedhydrolysate and post-hydolysate comprising both C5 and C6 monomer sugarsderived from both cellulose and hemicellulose can be directly fermentedto ethanol by modified yeast.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows xylan number as a function of pretreatment severity factorfor soft lignocellulosic biomass feedstocks subject to autohydrolysispretreatment. Soft lignocellulosic biomass feedstocks in the figureinclude pretreated wheat straw (PWS), corn stover (PCS), sugarcanebagasse (SCB) and empty fruit bunches from oil palm (PEFB).

FIG. 2 shows C5 recovery in soluble and insoluble form as a function ofxylan number for soft lignocellulosic biomass feedstocks subject toautohydrolysis pretreatment. Specifically, FIG. 2 shows the recovery oforiginal hemicellulose sugars from wheat straw after pretreatmentexpressed as water insoluble solids (WIS) and water soluble solids (WSS)as a function of Xylan number.

FIG. 3 shows total C5 recovery as a function of xylan number for softlignocellulosic biomass feedstocks subject to autohydrolysispretreatment. Specifically FIG. 3 shows the recovery of originalhemicellulose sugars after pretreatment as a function of Xylan numberfor wheat straw (PWS), corn stover (PCS), sugarcane bagasse (PSCB) andempty fruit bunches (PEFB).

FIG. 4 shows production of acetic acid, furfural and5-(hydroxymethyl)furfural (5-HMF) as a function of xylan number for softlignocellulosic biomass feedstocks subject to autohydrolysispretreatment.

FIG. 5 shows the effect of removal of dissolved solids on celluloseconversion for soft lignocellulosic biomass feedstocks subject to verylow severity autohydrolysis pretreatment.

FIG. 6 shows high performance liquid chromatography (HPLC)characterization of liquid fraction from soft lignocellulosic biomassfeedstocks subject to very low severity autohydrolysis pretreatment.Specifically, FIG. 6 shows HPLC characterization of liquid fraction fromwheat straw pretreated by Autohydrolysis to xylan number 11.5%.

FIG. 7 shows C5 sugar recovery as a function of time where solidfraction is subject to enzymatic hydrolysis followed by introduction ofliquid fraction for post-hydrolysis. Specifically, FIG. 7 shows thehydrolysis profile showing conversions of C5 sugars to monomers duringhydrolysis of solid fraction and after addition of liquid fraction at 96hours expressed as % theoretical yield.

FIG. 8 shows fermentation profile of ethanol fermentation by a modifiedyeast strain using wheat straw that was pretreated by very low severityautohydrolysis, enzymatically hydrolysed and used as combined liquid andsolid fraction without de-toxification to remove fermentationinhibitors. Specifically, FIG. 8 shows the fermentation by yeast strainV1 from Terranol of steam pretreated wheat straw (xylan number >10%)that was previously hydrolyzed by Celic Ctec2 from Novozymes and used ascombined liquid and solid fraction without de-toxification to removefermentation inhibitors.

FIG. 9 shows a process scheme for one embodiment, wherein theabbreviation DM refers to dry matter.

DETAILED DESCRIPTION OF EMBODIMENTS

In some embodiments the invention provides methods of processinglignocellulosic biomass comprising:

-   -   Providing soft lignocellulosic biomass feedstock,    -   Pretreating the feedstock at pH within the range 3.5 to 9.0 in a        single-stage pressurized hydrothermal pretreatment to very low        severity such that the pretreated biomass is characterized by        having a xylan number of 10% or higher,    -   Separating the pretreated biomass into a solid fraction and a        liquid fraction,    -   Hydrolysing the solid fraction with or without addition of        supplemental water content using enzymatic hydrolysis catalysed        by an enzyme mixture comprising endoglucanase, exoglucanase,        B-glucosidase, endoxylanase, xylosidase and acetyl xylan        esterase activities, and    -   Subsequently mixing the separated liquid fraction and the        hydrolysed solid fraction, whereby xylo-oligomers in the liquid        fraction are degraded to xylose monomers by the action of enzyme        activities remaining within the hydrolysed solid fraction.

As used herein, the following terms have the following meanings:

“About” as used herein with reference to a quantitative number or rangerefers to +/−10% in relative terms of the number or range referred to.

“Autohydrolysis” refers to a pretreatment process in which acetic acidliberated by hemicellulose hydrolysis during pretreatment furthercatalyzes hem icellulose hydrolysis, and applies to any hydrothermalpretreatment of lignocellulosic biomass conducted at pH between 3.5 and9.0.

“Commercially available cellulase preparation optimized forlignocellulosic biomass conversion ” refers to a commercially availablemixture of enzyme activities that is sufficient to provide enzymatichydrolysis of pretreated lignocellulosic biomass and that comprisesendocellulase (endoglucanase), exocellulase (exoglucanase),endoxylanase, acetyl xylan esterase, xylosidase and B-glucosidaseactivities. The term “optimized for lignocellulosic biomass conversion”refers to a product development process in which enzyme mixtures havebeen selected and/or modified for the specific purpose of improvinghydrolysis yields and/or reducing enzyme consumption in hydrolysis ofpretreated lignocellulosic biomass to fermentable sugars.

Conducting pretreatment “at” a dry matter level refers to the dry mattercontent of the feedstock at the start of pressurized hydrothermalpretreatment. Pretreatment is conducted “at” a pH where the pH of theaqueous content of the biomass is that pH at the start of pressurizedhydrothermal pretreatment.

“Dry matter,” also appearing as DM, refers to total solids, both solubleand insoluble, and effectively means “non-water content.” Dry mattercontent is measured by drying at 105° C. until constant weight isachieved.

“Fiber structure” is maintained to the extent that the average size offiber fragments following pretreatment is >750 um.

“Hydrothermal pretreatment” refers to the use of water, either as hotliquid, vapor steam or pressurized steam comprising high temperatureliquid or steam or both, to “cook” biomass, at temperatures of 120° C.or higher, either with or without addition of acids or other chemicals.

“Single-stage pressurized hydrothermal pretreatment” refers to apretreatment in which biomass is subject to pressurized hydrothermalpretreatment in a single reactor configured to heat biomass in a singlepass and in which no further pressurized hydrothermal pretreatment isapplied following a solid/liquid separation step to remove liquidfraction from feedstock subject to pressurized hydrothermalpretreatment.

“Solid/liquid separation” refers to an active mechanical process wherebyliquid is separated from solid by application of force through pressing,centrifugal or other force.

“Soft lignocellulosic biomass” refers to plant biomass other than woodcomprising cellulose, hemicellulose and lignin.

“Solid fraction” and “Liquid fraction” refer to fractionation ofpretreated biomass in solid/liquid separation. The separated liquid iscollectively referred to as “liquid fraction.”

The residual fraction comprising considerable insoluble solid content isreferred to as “solid fraction.” A “solid fraction” will have a drymatter content and typically will also comprise a considerable residualof “liquid fraction.”

“Theoretical yield” refers to the molar equivalent mass of pure monomersugars obtained from polymeric cellulose, or from polymeric hemicellulose structures, in which constituent monomeric sugars may also beesterified or otherwise substituted. “C5 monomer yields” as a percentageof theoretical yield are determined as follows: Prior to pretreatment,biomass feedstock is analysed for carbohydrates using the strong acidhydrolysis method of Sluiter et al. (2008) using an HPLC column andelution system in which galactose and mannose co-elute with xylose.Examples of such systems include a REZEX™ Monossacharide H+column fromPhenomenex and an AMINEX HPX 87C™ column from Biorad. During strong acidhydrolysis, esters and acid-labile substitutions are removed. Except asotherwise specified, the total quantity of “Xylose” +Arabinosedetermined in the un-pretreated biomass is taken as 100% theoretical C5monomer recovery, which can be termed collectively “C5 monomerrecovery.” Monomer sugar determinations are made using HPLCcharacterization based on standard curves with purified externalstandards. Actual C5 monomer recovery is determined by HPLCcharacterization of samples for direct measurement of C5 monomers, whichare then expressed as a percent of theoretical yield.

“Xylan number” refers to a characterization of pretreated biomassdetermined as follows: Pretreated biomass is subject to solid/liquidseparation to provide a solid fraction at about 30% total solids and aliquid fraction. This solid fraction is then partially washed by mixingwith 70° C. water in the ratio of total solids (DM) to water of 1:3wt:wt. The solid fraction washed in this manner is then pressed to about30% total solids. Xylan content of the solid fraction washed in thismanner is determined using the method of A. Sluiter, et al.,“Determination of structural carbohydrates and lignin in biomass,” USNational Renewable Energy Laboratory (NREL) Laboratory AnalyticalProcedure (LAP) with issue date Apr. 25, 2008, as described in TechnicalReport NREL/TP-510-42618, revised April 2008. An HPLC column and elutionsystem is used in which galactose and mannose co-elute with xylose.Examples of such systems include a REZEX™ Monossacharide H+ column fromPhenomenex and an AMINEX HPX 87C™ column from Biorad. This measurementof xylan content as described will include some contribution of solublematerial from residual liquid fraction that is not washed out of solidfraction under these conditions. Accordingly, “xylan number” provides a“weighted combination” measurement of residual xylan content withininsoluble solids and of soluble xylose and xylo-oligomer content withinthe “liquid fraction.”

Any suitable soft lignocellulosic biomass may be used, includingbiomasses such as at least wheat straw, corn stover, corn cobs, emptyfruit bunches, rice straw, oat straw, barley straw, canola straw, ryestraw, sorghum, sweet sorghum, soybean stover, switch grass, Bermudagrass and other grasses, bagasse, beet pulp, corn fiber, or anycombinations thereof. Lignocellulosic biomass may comprise otherlignocellulosic materials such as paper, newsprint, cardboard, or othermunicipal or office wastes. Lignocellulosic biomass may be used as amixture of materials originating from different feedstocks, may befresh, partially dried, fully dried or any combination thereof. In someembodiments, methods of the invention are practiced using at least about10 kg biomass feedstock, or at least 100 kg, or at least 500 kg.

Lignocellulosic biomass comprises crystalline cellulose fibrilsintercalated within a loosely organized matrix of hemicellulose andsealed within an environment rich in hydrophobic lignin. While celluloseitself comprises long, straight chain polymers of D-glucose,hemicellulose is a heterogeneous mixture of short, branched-chaincarbohydrates including monomers of all the 5-carbon aldopentoses (C5sugars) as well as some 6-carbon (C6) sugars including glucose andmannose. Lignin is a highly heterogeneous polymer, lacking anyparticular primary structure, and comprising hydrophobic phenylpropanoidmonomers.

Suitable lignocellulosic biomass typically comprises cellulose inamounts between 20 and 50% of dry mass prior to pretreatment, lignin inamounts between 10 and 40% of dry mass prior to pretreatment, andhemicellulose in amounts between 15 and 40%.

In some embodiments, biomass feedstocks may be subject to particle sizereduction and/or other mechanical processing such as grinding, milling,shredding, cutting or other processes prior to hydrothermalpretreatment. In some embodiments, biomass feedstocks may be washedand/or leached of valuable salts prior to pressurized pretreatment, asdescribed in Knudsen et al. (1998). In some embodiments feedstocks maybe soaked prior to pressurized pretreatment at temperatures up to 99° C.

In some embodiments the feedstock is first soaked in an aqueous solutionprior to hydrothermal pretreatment. In some embodiments, it can beadvantageous to soak the feedstock in an acetic acid containing liquidobtained from a subsequent step in the pretreatments, as described inU.S. Pat. No. 8,123,864. It is advantageous to conduct treatment at thehighest possible dry matter content, as described in U.S. Ser. No.12/935,587. Conducting pretreatment at high dry matter avoidsexpenditure of process energy on heating of unnecessary water. However,some water content is required to achieve optimal eventual sugar yieldsfrom enzymatic hydrolysis. Typically it is advantageous to pretreatbiomass feedstocks at or close to their inherent water holding capacity.This is the level of water content that a given feedstock will attainafter soaking in an excess of water followed by pressing to themechanical limits of an ordinary commercial screw press—typicallybetween 30 and 45% DM. In some embodiments, hydrothermal pretreatment isconducted at DM content at least 35%. It will be readily understood byone skilled in the art that DM content may decrease during hydrothermalpretreatment as some water content is added during heating. In someembodiments, feedstocks are pretreated at DM content at least 20%, or atleast 25%, or at least 30%, or at least 40%, or 40% or less, or 35% orless, or 30% or less.

In some embodiments, soaking/wetting with an aqueous solution can serveto adjust pH prior to pretreatment to the range of between 3.5 and 9.0,which is typically advantageous for autohydrolysis. It will be readilyunderstood that pH may change during pretreatment, typically to moreacidic levels as acetic acid is liberated from solubilizedhemicellulose.

In some embodiments, hydrothermal pretreatment is conducted withoutsupplemental oxygen as required for wet oxidation pretreatments, orwithout addition of organic solvent as required for organosolvpretreatment, or without use of microwave heating as required formicrowave pretreatments. In some embodiments, hydrothermal pretreatmentis conducted at temperatures of 140° C. or higher, or at 150° C. orhigher, or at 160° C. or higher, or between 160 and 200° C., or between170 and 190° C., or at 180° C. or lower, or at 170° C. or lower.

In some embodiments, some C5 content may be removed by a soaking stepprior to pressurized pretreatment. In some embodiments, the singlereactor may be configured to heat biomass to a single targettemperature. Alternatively, the single reactor may be configured toaffect a temperature gradient within the reactor such that biomass isexposed, during a single passage, to more than one temperature region.In some embodiments, it may be advantageous to partially remove somesolubilized biomass components from within the pressurized reactorduring the course of pretreatment.

Suitable hydrothermal pretreatment reactors typically include mostpulping reactors known from the pulp and paper industry. In someembodiments, hydrothermal pretreatment is administered by steam within areactor pressurized to 10 bar or lower, or to 12 bar or lower, or to 4bar or higher, or 8 bar or higher, or between 8 and 18 bar, or between18 and 20 bar. In some embodiments, the pretreatment reactor isconfigured for a continuous inflow of feedstock.

In some embodiments, wetted biomass is conveyed through the reactor,under pressure, for a certain duration or “residence time.” Residencetime is advantageously kept brief to facilitate higher biomassthroughput. However, the pretreatment severity obtained is determinedboth by temperature and also by residence time. Temperature duringhydrothermal pretreatment is advantageously kept lower, not only becausemethods of the invention seek to obtain a very low pretreatmentseverity, but also because lower temperatures can be accomplished usinglower steam pressures. To the extent that pretreatment temperature canbe at levels of 180° C. or lower, and accordingly, saturated steampressures kept to 10 bar or lower, lower tendency for corrosion isexperienced and much lower grade pressure fittings and steelcompositions may be used, which reduces plant capital costs. In someembodiments, the reactor is configured to heat biomass to a singletarget temperature between 160 and 200° C., or between 170 and 190° C.Residence times in some embodiments are less than 60, or less than 30,or less than 20, or less than 15, or less than 14, or less than 13, orless than 12, or less than 10, or less than 8, or less than 5 minutes.

Biomass feedstocks may be loaded from atmospheric pressure into apressurized reactor by a variety of means. In some embodiments, asluice-type “particle pump” system may be used to load biomassfeedstocks, such as the system described in U.S. Ser. No. 13/062,522. Insome embodiments, it may be advantageous to load a pretreatment reactorusing a so-called “screw plug” feeder.

Pretreated biomass may be unloaded from a pressurized reactor by avariety of means. In some embodiments, pretreated biomass is unloaded insuch manner as to preserve the fiber structure of the material.Preserving the fiber structure of the pretreated biomass is advantageousbecause this permits the solid fraction of the pretreated material to bepressed during solid/liquid separation to comparatively high dry matterlevels using ordinary screw press equipment, and thereby avoiding theadded expense and complexity of membrane filter press systems.

Fiber structure can be maintained by removing the feedstock from thepressurized reactor in a manner that is non-explosive. In someembodiments, non-explosive removal may be accomplished and fiberstructure thereby maintained using a sluice-type system, such as thatdescribed in U.S. Ser. No. 13/043,486. In some embodiments,non-explosive removal may be accomplished and fiber structure therebymaintained using a hydrocyclone removal system, such as those describedin U.S. Ser. No. 12/996,392.

In some embodiments, pretreated biomass can be removed from apressurized pretreatment reactor using “steam explosion,” which involvesexplosive release of the pretreated material. Steam-exploded, pretreatedbiomass does not retain its fiber structure and accordingly requiresmore elaborate solid/liquid separation systems in order to achieve drymatter content comparable to that which can be achieved using ordinaryscrew press systems with pretreated biomass that retains its fiberstructure.

The biomass feedstock is pretreated to very low severity, such that thepretreated biomass is characterized by having a xylan number of 10% orhigher. In some embodiments, the biomass is pretreated to a xylan numberof 11% or higher, or 12% or higher, or 13% or higher, or 14% or higher,or 15% or higher, or 16% or higher, or 17% or higher. The parameter“xylan number” refers to a composite measurement that reflects aweighted combination of both residual xylan content remaining withininsoluble solids and also the concentration of soluble xylose andxylo-oligomers within the liquid fraction. At lower Ro severity, xylannumber is higher. Thus, the highest xylan number refers to the lowestpretreatment severity. Xylan number provides a negative linearcorrelation with the conventional severity measure log R₀ even to verylow severity, where residual xylan content within insoluble solids is10%or higher.

Xylan number is particularly useful as a measure of pretreatmentseverity in that different pretreated biomass feedstocks havingequivalent xylan number exhibit equivalent C5 monomer recovery. Incontrast, conventional Ro severity is simply an empirical description ofpretreatment conditions, which does not provide a rational basis forcomparisons between different biomass feedstocks. For example,single-stage autohydrolysis to severity log R₀=3.75 provides pretreatedsugar cane bagasse and corn stover having a xylan number of between6-7%, while with typical wheat straw strains, the resulting xylan numberof pretreated feedstock is about 10%.

It is advantageous that biomass feedstocks be pretreated to very lowseverity wherein xylan number of the pretreated feedstock is 10% orgreater. This very low severity level corresponds to a process in whichthe total hemicellulose content of the feedstock before pretreatmentthat is either solubilized or irretrievably lost during pretreatment isminimized. At xylan number 10% and higher, with typical strains of wheatstraw, sugar cane bagasse, sweet sorghum bagasse, corn stover, and emptyfruit bunches (from oil palm), at least 60% of the original C5 contentof the feedstock can be recovered after single-stage autohydrolysispretreatment, where both xylan in the solid fraction and also solublexylose and xylo-oligomers in the liquid fraction are accounted for. Wehave unexpectedly discovered that high final C5 monomer yields of atleast 55% theoretical, or at least 60%, or at least 65%, can be obtainedwithout appreciable loss of C6 monomer yields after enzymatic hydrolysisof feedstocks pretreated to very low severity by single-stageautohydrolysis. At very low severity levels, a large fraction of thefeedstock's hem icellulose content remains within the solid fractionafter pretreatment, where it can subsequently be hydrolysed to C5monomers with high recovery using enzymatic hydrolysis.

It should be noted that reports concerning “xylose recovery” are oftenexpressed in terms that are not comparable to the xylose recoveriesreported here. For example, Ohgren et al. (2007) and Lee et al. (2009)report high xylose recoveries. But these values refer only to xyloserecovery from pretreated biomass, not expressed as a percentage of theoriginal hemicellulose content of the feedstock prior to pretreatment.Or for example WO2010/113129 refers to hemicellulose recovery as apercentage of hemicellulose content of the feedstock prior topretreatement, but does not specify the monomer yield, which isinvariable smaller than the total hemicellulose recovery.

Another startling feature of biomass that has been pretreated bysingle-stage autohydrolysis to very low severity levels is that theconcentrations of pretreatment by-products that serve as inhibitors offermentive organisms are kept to very low levels. As a consequence, itis typically possible to use hydrolysed biomass obtained by methods ofthe invention directly in fermentations, without requirement for anywashing or other de-toxification step.

As is well known in the art, autohydrolysis hydrothermal pretreatmenttypically produces a variety of soluble by-products which act as“fermentation inhibitors,” in that these inhibit growth and/ormetabolism of fermentive organisms. Different fermentation inhibitorsare produced in different amounts, depending on the properties of thelignocellulosic feedstock and on the severity of pretreatment. SeeKlinke et al. (2004). At least three categories of fermentationinhibitors are typically formed during autohydrolysis pretreatment: (1)furans, primarily 2-furfural and 5-HMF (5 hydroxymethylfurfural) whichare degradation products from mono- or oligo-saccharides; (2) monomericphenols, which are degradation products of the lignin structure; and (3)small organic acids, primarily acetic acid, which originate from acetylgroups in hem icelluloses, and lignin. The mixture of differentinhibitors has been shown to act synergistically in bioethanolfermentation using yeast strains, see e.g. Palmquist et al. (1999), and,also, using ethanolic Escherichia coli, see e.g. Zaldivar et al. (1999).In some embodiments, it can be advantageous to subject pretreatedbiomass to flash evaporation, using methods well known in the art, inorder to reduce levels of volatile inhibitors, most notably furfural.Using autohydrolysis with typical strains of biomass feedstocks such aswheat straw, sweet sorghum bagasse, sugar cane bagasse, corn stover, andempty fruit bunches, pretreated to xylan number 10% or higher, in ourexperience only acetic acid and furfural levels are potentiallyinhibitory of fermentive organisms. Where biomass feedstocks arepretreated at DM 35% or higher to xylan number 10% or higher, and wheresolid fraction is subsequently hydrolysed enzymatically at 25% or lowerDM, with added water to adjust DM but without washing steps, furfurallevels in the hydrolysate can typically be kept under 3 g/kg and aceticacid levels beneath 9 g/kg. These levels are typically acceptable foryeast fermentations using specialized strains. During enzymatichydrolysis, some additional acetic acid is released from degradation ofhem icellulose in the solid fraction. In some embodiments, it may beadvantageous to remove some acetic acid content from liquid fractionand/or hydrolysed solid fraction using electrodialysis or other methodsknown in the art.

Different feedstocks can be pretreated using single-stage autohydrolysisto xylan number 10% or greater by a variety of different combinations ofreactor residence times and temperatures. One skilled in the art willreadily determine through routine experimentation an appropriatepretreatment routine to apply with any given feedstock, using any givenreactor, and with any given biomass reactor-loading andreactor-unloading system. Where feedstocks are pretreated using acontinuous reactor, loaded by either a sluice-system or a screw-plugfeeder, and unloaded by either a “particle pump” sluice system or ahydrocyclone system, very low severity of 10% or greater xylan numbercan be achieved using typical strains of wheat straw or empty fruitbunches by a temperature of 180° C. and a reactor residence time of 24minutes. For typical strains of corn stover, sugar cane bagasse, andsweet sorghum bagasse, very low severity of 10% or greater xylan numbercan typically be achieved using a temperature of 180° C. and a reactorresidence time of 12 minutes, or using a temperature of 175° C. and areactor residence time of 17 minutes. It will be readily understood byone skilled in the art that residence times and temperatures maybeadjusted to achieve comparable levels of R₀ severity.

Following pretreatment, pretreated biomass is separated into a solidfraction and a liquid fraction by a solid/liquid separation step. Itwill be readily understood that “solid fraction” and “liquid fraction”may be further subdivided or processed. In some embodiments, biomass maybe removed from a pretreatment reactor concurrently with solid/liquidseparation. In some embodiments, pretreated biomass is subject to asolid/liquid separation step after it has been unloaded from thereactor, typically using a simple and low cost screw press system, togenerate an solid fraction and a liquid fraction. Cellulase enzymeactivities are inhibited by liquid fraction, most notably due toxylo-oligomer content but possibly also due to phenol content and/orother compounds not yet identified. It is accordingly advantageous toachieve the highest practicable levels of dry matter content in thesolid fraction or, alternatively, to remove the highest practicableamount of dissolved solids from the solid fraction. In some embodiments,solid/liquid separation achieves a solid fraction having a DM content ofat least 40%, or at least 45%, or at least 50% , or at least 55%.Solid/liquid separation using ordinary screw press systems can typicallyachieve DM levels as high as 50% in the solid fraction, provided thebiomass feedstock has been pretreated in such manner that fiberstructure is maintained. In some embodiments, it may be advantageous toincur higher plant capital expenses in order to achieve more effectivesolid/liquid separation, for example, using a membrane filter presssystem. In some embodiments, dissolved solids can be removed from asolid fraction by serial washing and pressing or by displacement washingtechniques known in the pulp and paper art. In some embodiments, eitherby solid/liquid separation directly, or by some combination of washingand solid/liquid separation, the dissolved solids content of the solidfraction is reduced by at least 50%, or at least 55%, or at least 60%,or at least 65%, or at least 70%, or at least 75%.

Enzymatic hydrolysis of feedstocks pretreated to xylan number 10% orhigher can typically be conducted with commercially reasonable enzymeconsumption, without requirement for specific washing or de-toxificationsteps, where the solid fraction is pressed to at least 40% DM, or wheredissolved solids content of the solid fraction is reduced by at least50%.

The liquid fraction obtained from solid/liquid separation is maintainedseparately from solid fraction during enzymatic hydrolysis of the solidfraction. We term this temporary separation “C5 bypass.” Liquid fractionobtained from soft lignocellulosic biomass feedstocks such as typicalstrains of wheat straw, sugar cane bagasse, sweet sorghum bagasse, cornstover, and empty fruit bunches pretreated by single-stageautohydrolysis to xylan number 10% or higher typically comprise a smallcomponent of C6 monomers (1x), primarily glucose with some other sugars;a larger component of soluble C6 oligomers (about 2x-7x); a largercomponent of C5 monomers (about 4x-8x), primarily xylose with somearabinose and other sugars; and a much larger component of solublexylo-oligomers (about 18x-30x). Soluble xylo-oligomers typically includeprimarily xylohexose, xylopentose, xylotetraose, xylotriose andxylobiose with some higher chain oligomers.

The solid fraction is subject to enzymatic hydrolysis using a mixture ofenzyme activities. As will be readily understood by one skilled in theart, the composition of enzyme mixtures suitable for practicing methodsof the invention may vary within comparatively wide bounds. Suitableenzyme preparations include commercially available cellulasepreparations optimized for lignocellulosic biomass conversion. Selectionand modification of enzyme mixtures during optimization may includegenetic engineering techniques, for example such as those described byZhang et al. (2006) or by other methods known in the art. Commerciallyavailable cellulase preparations optimized for lignocellulosic biomassconversion are typically identified by the manufacturer and/or purveyoras such. These are typically distinct from commercially availablecellulase preparations for general use or optimized for use inproduction of animal feed, food, textiles detergents or in the paperindustry. In some embodiments, a commercially available cellulasepreparation optimized for lignocellulosic biomass conversion is usedthat is provided by GENENCOR™ and that comprises exoglucanases,endoglucanases, endoxylanases, xylosidases, acetyl xylan esterases andbeta glucosidases isolated from fermentations of genetically modifiedTrichoderma reesei, such as, for example, the commercial cellulasepreparation sold under the trademark ACCELLERASE TRIO™. In someembodiments, a commercially available cellulase preparation optimizedfor lignocellulosic biomass conversion is used that is provided byNOVOZYMES™ and that comprises exoglucanases, endoglucanases,endoxylanases, xylosidases, acetyl xylan esterases and betaglucosidases, such as, for example, the commercial cellulasepreparations sold under either of the trademarks CELLIC CTEC2™ or CELLICCTEC3™.

The enzyme activities represented in three commercially availablecellulase preparation optimized for lignocellulosic biomass conversionwere analysed in detail. Each of these three preparations, ACCELLERASETRIO™ from GENENCOR™ and CELLIC CTEC2™ and CELLIC CTEC3™ fromNOVOZYMES™, was shown to be effective at enzyme dose levels within themanufacturers' suggested range, in providing combined C5/C6 wheat strawhydrolysate prepared according to methods of the invention in which C5monomer yields were at least 60% and cellulose C6 conversion yields wereat least 60%. For each of these commercial cellulase preparations,levels of twelve different enzyme activities were characterized andexpressed per gram protein. Experimental details are provided in Example8. Results are shown in Table 1.

TABLE 1 Selected activity measurements in commercial cellulasepreparations optimized for lignocellulosic biomass conversion. ActivityUnit definition CTEC3 ACTrio CTEC2 Substrate (formation) CBH I 454 ± 2.5U/g 171 ± 0.4 U/g 381 ± 21 U/g MeUmb-3- cellobioside 1 μmole MeUmdequivalent/min CBH II* Not measurable Not measurable Not measurableEndo-1,4-β-glucanase 466 ± 31 U/g 149 ± 21 U/g 173 ± 15 U/g AvicelPH-101 1 μmole glucose equivalent/min. β-glucosidase 3350 ± 75 U/g 891 ±60 U/g 2447 ± 70 U/g Cellobiose 2 μmole glucose/min. (Conversion of 1μmole cellobiose/min) Endo-1,4-β-xylanase 278 ± 10 U/g 799 ± 55 U/g 306± 41 U/g WEAX (medium visc.) 1 μmole glucose equivalent/min.β-xylosidase 279 ± 7.0 U/g U/g 431 ± 22 U/g 87 ± 0.2 U/g WEAX (mediumvisc.) 1 μmole xylose/min. β-L-arabinofuranosidase 20 ± 1.0 U/g U/g 9.4± 0.4 U/g 12 ± 0.1 U/g WEAX (medium visc.) 1 μmole arabinose/min.Laccase No activity No activity No activity Syringaldazine —Amyloglucosidase (AMG) 18 ± 3.6 U/g U/g 29 ± 0.1 U/g 18 ± 1.5 U/g Cornstarch (soluble) 1 μmole glucose/min. o-amylase 2.7 ± 0.1 U/g U/g 3.4 ±0.5 U/g 4.7 ± 1.4 U/g Corn starch (soluble) 1 μmole glucoseequivalent/min. Acetyl xylan esterase 3.8 · 10⁻³ ± 9 · 3.1 · 10⁻⁴ ± 1 ·4.2 · 10⁻³ ± 4.2 · pNP-acetate 1 μmole pNP equivalent min. 10⁻⁵ U/g 10⁻⁴U/g 10⁻⁴ U/g Ferulic acid esterase No activity No activity No activityMethyl ferulate —

In some embodiments, enzyme preparations may be used that have similarrelative proportions as those exhibited by the commercial preparationsdescribed in Table 1 between any of the endoglucanase, exoglucanase,B-glucosidase, endoxylanase, xylosidase and/or acetyl xylan esteraseactivities.

Enzyme mixtures that are effective to hydrolyse lignocellulosic biomasscan alternatively be obtained by methods well known in the art from avariety of microorganisms, including aerobic and anaerobic bacteria,white rot fungi, soft rot fungi and anaerobic fungi. See e.g. Singhaniaet al. (2010). Organisms that produce cellulases typically secrete amixture of different enzymes in appropriate proportions so as to besuitable for hydrolysis of lignocellulosic substrates. Preferred sourcesof cellulase preparations useful for conversion of lignocellulosicbiomass include fungi such as species of Trichoderma, Penicillium,Fusarium, Humicola, Aspergillus and Phanerochaete.

One fungus species in particular, Trichoderma reesei, has beenextensively studied. Wild type Trichoderma reesei secretes a mixture ofenzymes comprising two exocellulases (cellobiohydrolases) withrespective specificities for reducing and non-reducing ends of cellulosechains, at least five different endocellulases having differingcellulose recognition sites, two B-glucosidases as well as a variety ofendoxylanases and exoxylosidases. See Rouvinen, J., et al. (1990);Divne, C., et al. (1994); Martinez, D., et al. (2008). Commercialcellulase preparations typically also include alpha-arabinofuranosidaseand acetyl xylan esterase activities. See e.g. Vinzant, T., et al.(2001).

An optimized mixture of enzyme activities in relative proportions thatdiffer from the proportions presented in mixtures naturally secreted bywild type organisms has previously been shown to produce higher sugaryields. See Rosgaard et al. (2007). Indeed, it is has been suggestedthat optimizations of enzyme blends including as many as 16 differentenzyme proteins can be advantageously determined separately for anygiven biomass feedstock subject to any given pretreatment. See Billard,H., et al. (2012); Banerjee, G., et al. (2010). As a commercialpracticality, however, commercial enzyme providers typically seek toproduce the smallest practicable number of different enzyme blends, inorder that economies of scale can be obtained in large-scale production.

In some embodiments, it can be advantageous to supplement a commerciallyavailable cellulase preparation optimized for lignocellulosic biomassconversion with one or more additional or supplemental enzymeactivities. In some embodiments, it may be advantageous simply toincrease the relative proportion of one or more component enzymespresent in the commercial preparation. In some embodiments, it may beadvantageous to introduce specialized additional activities. Forexample, in practicing methods of the invention using any given biomassfeedstock, particular unhydrolysed carbohydrate linkages may beidentified that could be advantageously hydrolysed through use of one ormore supplemental enzyme activities. Such unhydrolysed linkages may beidentified through characterization of oligomeric carbohydrates, usingmethods well known in the art, in soluble hydrolysates or in insolubleunhydrolysed residual. Unhydrolysed linkages may also be identifiedthrough comprehensive microarray polymer profiling, using monoclonalantibodies directed against specific carbohydrate linkages, as describedby Nguema-Ona et al. (2012). In some embodiments it can be advantageousto supplement a commercially available cellulase preparation optimizedfor lignocellulosic biomass conversion using any one or more ofadditional endoxylanase, B-glucosidase, mannanase, glucouronidase, xylanesterase, amylase, xylosidase, glucouranyl esterase, orarabinofuranosidase.

In some embodiments, it can alternatively be advantageous to produceenzymes on-site at a lignocellulosic biomass processing facility, asdescribed by Humbird et al. (2011). In some embodiments, a commerciallyavailable cellulase preparation optimized for lignocellulosic biomassconversion may be produced on-site, with or without customizedsupplementation of specific enzyme activities appropriate to aparticular biomass feedstock.

In some embodiments, whether or not a commercially available cellulasepreparations optimized for lignocellulosic biomass conversion is used,and whether or not enzymes are produced on-site at a biomass processingplant, advantages of the invention can be obtained using softlignocellulosic biomass feedstocks subject to autohydrolysispretreatment to very low severity xylan number 10% or greater using anenzyme mixture that comprises the following: (1) Exocellulase(cellobiohydrolase) activities (EC 3.2.1.91), optionally including atleast two enzymes with specificities for reducing and non-reducing endsof cellulose chains; (2) endocellulase activity (EC 3.2.1.4); (3)B-glucosidase activity

(EC 3.2.1.21); (4) B-1,4 endoxylanase activity (EC 3.2.1.8); (5) acetylxylan esterase activity (EC 3.1.1.72); and optionally (6) B-1,3xylosidase activity (EC 3.2.1.72); and optionally (7) B-1,4 xylosidaseactivity (EC 3.2.1.37); and optionally (8) alpha 1,3 and/or alpha 1, 5arabinofuranosidase activity (EC 3.2.1.23). In some embodiments, theenzyme mixture is further characterized by having relative proportionsof enzyme activities as follows: 1 FPU cellulase activity is associatedwith at least 30 CMC U endoglucanase activity and with at least at least28 pNPG U beta glucosidase activity and with at least 50 ABX Uendoxylanase activity. It will be readily understood by one skilled inthe art that CMC U refers to carboxymethycellulose units, where one CMCU of activity liberates 1 umol of reducing sugars (expressed as glucoseequivalents) in one minute under specific assay conditions of 50° C. andpH 4.8; that pNPG U refers to pNPG units, where one pNPG U of activityliberates 1 umol of nitrophenol per minute frompara-nitrophenyl-B-D-glucopyranoside at 50° C. and pH 4.8; and that ABXU refers to birchwood xylanase units, where one ABX U of activityliberates 1 umol of xylose reducing sugar equivalent in one minute at50° C. and pH 5.3. It will be further readily understood by one skilledin the art that FPU refers to “filter paper units,” which provides ameasure of total cellulase activity including any mixture of differentcellulase enzymes. As used herein, FPU refers to filter paper units asdetermined by the method of Adney, B. and Baker, J., LaboratoryAnalytical Procedure #006, “Measurement of cellulase activity”, August12, 1996, the USA National Renewable Energy Laboratory (NREL).

In some embodiments the enzyme mixture may further include any one ormore of mannosidases (EC 3.2.1.25), a-D-galactosidases (EC 3.2.1.22),a-L-arabinofuranosidases (EC 3.2.1.55), a-D-glucuronidases (EC3.2.1.139), cinnamoyl esterases (EC 3.1.1.-), or feruloyl esterases (EC3.1.1.73).

One skilled in the art will readily determine, through routineexperimentation, an appropriate dose level of any given enzymepreparation to apply, and an appropriate duration for enzymatichydrolysis. It is generally advantageous to maintain lower enzyme doselevels, so as to minimize enzyme costs. In some embodiments, it can beadvantageous to use a high enzyme dose. In practicing methods of theinvention, one skilled in the art can determine an economic optimisationof enzyme dose in consideration of relevant factors including localbiomass costs, market prices for product streams, total plant capitalcosts and amortization schemes, and other factors. In embodiments wherea commercially available cellulase preparation optimized forlignocellulosic biomass conversion is used, a general dose rangeprovided by manufacturers can be used to determine the general rangewithin which to optimize. Hydrolysis duration in some embodiments is atleast 48 hours, or at least 64 hours, or at least 72 hours, or at least96 hours, or for a time between 24 and 150 hours.

As is well known in the art, cellulase catalysis is more efficient wherehydrolysis is conducted at low dry matter content. Higher solidsconcentration effectively inhibits cellulase catalysis, although theprecise reasons for this well known effect are not fully understood. Seee.g. Kristensen et al. (2009).

In some embodiments, it may be advantageous to conduct hydrolysis atvery high DM>20%, notwithstanding some resulting increase in enzymeconsumption. It is generally advantageous to conduct hydrolysis at thehighest practicable dry matter level, both in order to minimize waterconsumption and waste water treatment requirements. It is additionallyadvantageous in fermentation systems to use the highest practicablesugar concentrations. Higher sugar concentrations are produced wherehydrolysis is conducted at higher dry matter levels. One skilled in theart will readily determine, through routine experimentation, a DM levelat which to conduct enzymatic hydrolysis that is appropriate to achievegiven process goals, for any given biomass feedstock and enzymepreparation. In some embodiments, enzymatic hydrolysis of the solidfraction may be conducted at 15% DM or greater, or at 16% DM or greater,or at 17% DM or greater, or at 18% DM or greater or at 19% DM orgreater, or at 20% DM or greater, or at 21% DM or greater, or at 22% DMor greater, or at 23% DM or greater, or at 25% DM or greater, or at 30%DM or greater, or at 35% DM or greater.

In some embodiments, solid fraction is recovered from solid/liquidseparation at 40% DM or greater, but additional water content is addedso that enzymatic hydrolysis may be conducted at lower DM levels. Itwill be readily understood that water content may be added in the formof fresh water, condensate or other process solutions with or withoutadditives such as polyethylene glycol (PEG) of any molecular weight orsurfactants, salts, chemicals for pH adjustment such as ammonia,ammonium hydroxide, calcium hydroxide, or sodium hydroxide,anti-bacterial or anti-fungal agents, or other materials.

After the solid fraction has been enzymatically hydrolysed to a desireddegree of conversion, the liquid fraction, which has been maintained inC5 bypass, is mixed with the hydrolysate mixture for post-hydrolysis. Insome embodiments, all of the recovered liquid fraction may be added atone time, while in other embodiments, some component of the liquidfraction may be removed and/or liquid fraction may be addedincrementally. In some embodiments, prior to mixing with liquidfraction, the solid fraction is hydrolysed to at least 50%, or at least55%, or at least 60% cellulose conversion, meaning that at least thespecified theoretical yield of glucose monomers is obtained. Asubstantial portion of xylo-oligomers present in liquid fraction cantypically be hydrolysed to xylose monomers by action of xylanase andother enzymes that remain active within the hydrolysate mixture. In someembodiments post-hydrolysis is conducted for at least 6 hours, or for atime between 15 and 50 hours, or for at least 24 hours. In someembodiments, at least 60%, or at least 65%, or at least 70%, or at least75%, or at least 80%, or at least 85%, or at least 90% by mass ofxylo-oligomers present in the liquid fraction are hydrolysed to xylosemonomers during post-hydrolysis by action of xylanase and other enzymesthat remain active within the hydrolysate mixture. In some embodiments,the liquid fraction is mixed with hydrolysate directly, without furtheraddition of chemical additives. In some embodiments, some components ofliquid fraction such as acetic acid, furfural or phenols may be removedfrom liquid fraction prior to mixing with hydrolysate.

In some embodiments, enzymatic hydrolysis of the solid fraction and/orpost-hydrolysis of the liquid fraction may be conducted as asimultaneous saccharification and fermentation (SSF) process. As is wellknown in the art, when SSF can be conducted at the same temperature asthat which is optimal for enzymatic hydrolysis, enzyme consumption canbe minimized because a fermentive organism introduced during the courseof enzymatic hydrolysis consumes glucose and xylose monomers and therebyreduces product inhibition of enzyme catalyzed reactions. In someembodiments, post-hydrolysis is only conducted after the fiber fractionhas been hydrolysed, without addition of fermentive organism, to atleast 60% cellulose conversion.

Where biomass feedstocks such as typical strains of wheat straw, sugarcane bagasse, sweet sorghum bagasse, corn stover or empty fruit bunchesare pretreated at 35% or greater DM by single-stage autohydrolysis toxylan number 10% or greater, where solid fraction of the pretreatedbiomass is obtained having at least 40% DM or having at least 50%removal of dissolved solids, where solid fraction is subsequentlysubject to enzymatic hydrolysis at DM between 15 and 27% using acommercially available cellulase preparation optimized forlignocellulosic biomass conversion, where enzymatic hydrolysis isconducted for at least 48 hours, where liquid fraction is added to thesolid fraction hydrolysate after at least 50% glucose conversion hasbeen obtained, and where the added liquid fraction is subject topost-hydrolysis for a period of at least 6 hours, it is typicallypossible to achieve C5 monomer concentrations in the combined C5/C6hydrolysate that correspond to C5 monomer yields of 60% or greater ofthe theoretical maximal xylose yield.

In some embodiments, the combined C5/C6 hydrolysate can be directlyfermented to ethanol using one or more modified yeast strains.

FIG. 9 shows a process scheme for one embodiment. As shown, softlignocellulosic biomass is soaked, washed or wetted to DM 35% orgreater. The biomass is pretreated at pH within the range of 3.5 to 9.0using pressurized steam in single-stage autohydrolysis to a severitycharacterized by xylan number 10% or greater. The pretreated biomass issubject to solid/liquid separation producing a liquid fraction and asolid fraction having DM content 40% or greater. The solid fraction isadjusted to an appropriate DM content then subject to enzymatichydrolysis at DM content 15% or greater to a degree of celluloseconversion 60% or greater. The separated liquid fraction is subsequentlymixed with the hydrolysed solid fraction and subject to post-hydrolysis,whereby a substantial quantity of xylo-oligomers present in the liquidfraction are hydrolysed to monomeric xylose. After the end of hydrolysisand post-hydrolysis as described, the C5 monomer yield is typically atleast 60% while the cellulose conversion is similarly at least 60%.

EXAMPLES Example 1 “Xylan Number” Characterization of Solid Fraction asa Measure of Pretreatment Severity

Wheat straw (WS), corn stover (CS) , Sweet sugarcane bagasse (SCB) andEmpty Fruit Bunches (EFB) were soaked with 0-10 g acetic acid/kg drymatter biomass, pH>4.0, prior to pretreatment at 35-50% dry matter about60 kg DM/h biomass was pretreated at temperatures from 170-200° C. witha residence time of 12-18 minutes. The biomass was loaded into thereactor using a sluice system and the pretreated material unloaded usinga sluice system. The pressure within the pressurized pretreatmentreactor corresponded to the pressure of saturated steam at thetemperature used. The pretreated biomass was subject to solid/liquidseparation using a screw press, producing a liquid fraction and a solidfraction having about 30% dry matter. The solid fraction was washed withabout 3 kg water/kg dry biomass and pressed to about 30% dry matteragain. Details concerning the pretreatment reactor and process arefurther described in Petersen et al. (2009).

Raw feedstocks were analysed for carbohydrates according to the methodsdescribed in Sluiter el al. (2005) and Sluiter et al. (2008) using aDionex Ultimate 3000 HPLC system equipped with a Rezex MonossacharideH+column from Phenomenex. Samples of liquid fraction and solid fractionwere collected after three hours of continuous pretreatment and sampleswere collected three times over three hours to ensure that a sample wasobtained from steady state pretreatment. The solid fractions wereanalysed for carbohydrates according to the methods described in Sluiteret al. (2008) with an Ultimate 3000 HPLC system from Dionex equippedwith a Rezex Monossacharide H+ Monosaccharide column. The liquidfractions were analysed for carbohydrates and degradation productsaccording to the methods described in Sluiter et al. (2006) with anUltimate 3000 HPLC system from Dionex equipped with a RezexMonossacharide H+Monosaccharide column. Degradation products in thesolid fraction were analysed by suspension of the solid fraction inwater with 5mM sulphuric acid in a ratio of 1:4 and afterward analysedaccording to the methods described in Sluiter et al. (2006) with anUltimate 3000 HPLC system from Dionex equipped with a RezexMonossacharide H+ column. The dry matter content and the amount ofsuspended solids was analysed according to the methods described inWeiss et al. (2009). Mass balances were set up as described in Petersenet al. (2009) and cellulose and hemicellulose recoveries weredetermined. The amount of sugars which were degraded to 5-HMF orfurfural and the amount of acetate released from hemicelleulose duringpretreatment per kg of biomass dry matter was quantified as well,although loss of furfural due to flashing is not accounted for.

The severity of a pretreatment process is commonly described by aseverity factor, first developed by Overend et al. (1987). The severityfactor is typically expressed as a log value such thatlog(R₀)=reksp((T-Tref)/14.75), where Ro is the severity factor, t is theresidence time in minutes, T is the temperature and Tref is thereference temperature, typically 100° C. The severity factor is based onkinetics of hemicellulose solubilisation as described by Belkecemi etal. (1991), Jacobsen and Wyman (2000) or Lloyd et al. (2003). Theseverity of a pretreatment is thus related to residual hemicellulosecontent remaining in the solid fraction after pretreatment.

Solid fractions prepared and washed as described were analysed for C5content according to the methods described by Sluiter et al. (2008) witha Dionex Ultimate 3000 HPLC system equipped with a Rezex MonossacharideH+ column from Phenomenex. The xylan content in the solid fractionproduced and washed as described above is linearly depended upon theseverity factor for soft lignocellulosic biomasses such as for examplewheat straw, corn stover of EFB when pretreating by hydrothermalautohydrolysis. The definition of severity as the xylan content in asolid fraction prepared and washed as described above is transferablebetween pretreatment setups. Xylan number is the measured xylan contentin the washed solid fractions, which includes some contribution fromsoluble material. The dependence of xylan number on pretreatmentseverity log(R₀) is shown in FIG. 1 for wheat straw, corn stover,sugarcane bagasse and empty fruit bunches from palm oil processing.

As shown, there exists a clear, negative linear correlation betweenxylan number and pretreatment severity for each of the tested biomassfeedstocks pretreated by single-stage autohydrolysis.

Example 2 C5 Recovery as a Function of Pretreatment Severity

Biomass feedstocks were pretreated and samples characterized asdescribed in example 1. FIG. 2 shows the C5 recoveries(xylose+arabinose) as a function of xylan number for experiments wherewheat straw was pretreated by autohydrolysis. C5 recoveries are shown aswater insoluble solids (WIS), water soluble solids (WSS) and totalrecovery. As shown, C5 recovery as both water insoluble and watersoluble solids increases as xylan number increases. As xylan numberincreases over 10%, C5 recovery as water soluble solids diminishes whileC5 recovery as water insoluble solids continues to increase

Typical strains of wheat straw tested contained about 27% hemicelluloseon dry matter basis prior to pretreatment. FIG. 3 shows total C5recovery after pretreatment as a function of xylan number for wheatstraw, corn stover, sugarcane bagasse and EFB pretreated byautohydrolysis. Typical strains of corn stover, sweet sugarcane bagasseand EFB tested contained about 25%, 19% and 23% respectively of C5content on dry matter basis prior to pretreatment. As shown, for allfeedstocks, total C5 recovery after pretreatment is dependent uponpretreatment severity as defined by xylan number. As shown, where 90% ofC5 content recovered after pretreatment can be fully hydrolysed to C5monomer, a 60% final C5 monomer yield after enzymatic hydrolysis can beexpected where pretreatment severity is characterized by producing axylan number of 10% or higher.

Example 3 Production of Degradation Products that Inhibit Enzymes andYeast Growth as a Function of Pretreatment Severity

Biomass feedstocks were pretreated and samples characterized asdescribed in example 1. FIG. 4 shows the dependence of acetic acidrelease and production of furfural and 5-hydroxy-methyl-fufural (5-HMF)as a function of xylan number for experiments where wheat straw waspretreated by single-stage autohydrolysis. As shown, production of thesedegradation products, which are well known to inhibit fermentive yeastand which in some cases also inhibit cellulase enzymes, exhibits anexponential increase at xylan numbers lower than 10%. At xylan number10% and higher, the levels of furfural and acetic acid fall withinranges that permit fermentation of pretreated biomass withoutrequirement for de-toxification steps. In the case of acetic acid,levels are further increased during enzymatic hydrolysis of biomasspretreated to xylan number 10% and higher, although typically to levelsthat are well tolerated by yeast modified to consume both C5 and C6sugars.

Example 4 Inhibition of Cellulase Enzymes by Material Remaining in SolidFraction as a Function of DM% of Solid Fraction

Experiments were conducted in a 6-chamber free fall reactor working inprinciple as the 6-chamber reactor described and used in WO2006/056838.The 6-chamber hydrolysis reactor was designed in order to performexperiments with liquefaction and hydrolysis at solid concentrationsabove 20% DM. The reactor consists of a horizontally placed drum dividedinto 6 separate chambers each 24 cm wide and 50 cm in height. Ahorizontal rotating shaft mounted with three paddles in each chamber isused for mixing/agitation. A 1.1 kW motor is used as drive and therotational speed is adjustable within the range of 2.5 and 16.5 rpm. Thedirection of rotation is programmed to shift every second minute betweenclock and anti-clock wise. A water-filled heating jacket on the outsideenables control of the temperature up to 80° C.

The experiments used wheat straw, pretreated by single-stageautohydrolysis. The biomass was wetted to a DM of >35% and pretreated atpH >4.0 by steam to xylan number 10.5%. . The pretreatment was conductedin the Inbicon pilot plant in Skrbk, Denmark. The biomass was loadedinto the pretreatment reactor using a sluice system and the pretreatedbiomass removed from the reactor using a sluice system. The pretreatedbiomass was, in some cases, subject to solid/liquid separation using ascrew press, producing a liquid fraction and a solid fraction. The solidfraction had a DM content of about 30%, contained the majority ofinitial cellulose and lignin, part of the hemicellulose and a total ofabout 25% of the dissolved solids.

The chambers of the 6 chamber reactor were filled with either totalpretreated biomass comprising all dissolved and undissolved solids orpressed solid fraction comprising about 25% of total dissolved solids.Dry matter content was adjusted to 19% DM. The pretreated biomass wasthen hydrolyzed at 50° C. and pH 5.0 to 5.3 using 0.08 ml CTec2™ fromNovozymes/g glucan or 0.2-0.3 ml Accellerase TRIO™ from Dupont,Genencor/g glucan. These dose levels of these commercially availablecellulase preparations optimized for lignocellulosic biomass conversionwere well within the range suggested by the manufacturers. Enzymatichydrolysis experiments were conducted for 96 hours at a mixing speed of6 rpm.

FIG. 5 shows cellulose conversion after enzymatic hydrolysis under theseconditions as a function of % dissolved solids removed prior toenzymatic hydrolysis. As shown, removal of 75% dissolved solids at theseenzyme dose levels improves cellulose conversion by 10-20% in absoluteterms. Thus, it is advantageous to press solid fraction to DM content atleast 40% or to otherwise reduce dissolved solids content by at least50% prior to enzymatic hydrolysis, since this will provide improvedenzyme performance.

Example 5 Sugar Content and Hydrolysis of Liquid Fraction from BiomassPretreated to Xylan Number >10%

Wheat straw, corn stover, and sugar cane bagasse were pretreated toxylan number 11.5% (WS), 12.3% (SCB) and 15.5% (CS) and subject tosolid/liquid separation to produce a liquid fraction and a solidfraction, as described in example 5. The liquid fractions were analysedfor carbohydrates and degradation products according to the methodsdescribed in (Sluiter, Hames et al. 2005) using a Dionex Ultimate 3000HPLC system equipped with a Rezex Monosaccharide column. Table 2 showsthe sugar content of liquid fractions expressed as a percent of DMcontent broken down into categories of oligomeric and monomericglucose/glucan, xylose/xylan and arabinose/arabinan. As shown, whilesome glucose content is present in both monomeric and oligomeric form,the bulk of the sugar content is oligomeric xylan. The predominance ofxylan oligomers in liquid fraction obtained using autohydrolysis is innoted contrast with the liquid fraction obtained using dilute acidpretreatment. In biomass pretreated by dilute acid hydrothermalpretreatment, the liquid fraction is typically hydrolysed to monomericconstituents by actions of the acid catalyst.

TABLE 2 Sugar content of liquid fractions in biomass pretreated to xylannumber > 10%. Oligo- Mono- Oligo- Mono- Oligo- Mono- meric meric mericmeric meric meric Other glucan glucose xylan xylose arabinan arabinoseDM WS 5.5% 2.1% 40.4% 8.6% 1.1% 4.8% 37% SCB 8.2% 3.1% 39.1% 8.7% 0.7%3.1% 37% SC 6.2% 1.9% 37.0% 5.3% 2.8% 3.9% 43%

The liquid fraction from pretreated wheat straw was furthercharacterized by HPLC analysis using a Thermo Scientific DionexCarboPac™ PA200 column using a modular Dionex ICS-5000 chromatographicsystem. The analytes were separated using NaOH/NaOAc-gradient conditionsand measured by integrated and pulsed amperometric detection (IPAD)using a gold electrode. FIG. 6 shows an HPLC chromatogram in which theelution profile of xylobiose (X2), xylotriose (X3), xylotetraose (X4),xylopentaose (X5), and xylohexaose (X6) standards is super-imposed asthe upper trace over the lower trace, which depicts the elution profileof liquid fraction. As shown, liquid fraction of the autohydrolysedbiomass contains a mixture comprising a small amount of xylose monomerand comparatively larger amounts of xylobiose (X2), xylotriose (X3),xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6), along withother materials.

Example 6 Enzymatic Hydrolysis of Solid Fraction and Addition of LiquidFraction After the Fibre Hydrolysis from Biomass Pretreated to XylanNumber >10% and Pressed to >40% DM Followed by Post Hydrolysis

Experiments were conducted in a 6-chamber free fall reactor as describedin example 4.

The experiments used wheat straw, corn stover, or sugar cane bagassepretreated by single-stage autohydrolysis to xylan numbers ranging from11.5 to 15.6%. The biomass was cut and wetted to a DM of >35% andpretreated by steam at 170-190° C. for 12 min. The pretreatment wasconducted in the Inbicon pilot plant in Skrbk, Denmark. The pretreatedbiomass was subject to solid/liquid separation using a screw press toproduce a solid fraction having >40% DM.

The chambers of the 6 chamber reactor were filled with about 10 kgpressed pretreated biomass and adjusted by water addition to 19-22% DM.The pretreated biomass was hydrolyzed at 50° C. and pH 5.0 to 5.3 usingACCELLERASE TRIO™ from GENENCOR-DuPONT. The mixing speed was 6 rpm. Thehydrolysis experiments were run for 96 hours and afterwards the liquidfraction pressed from the solid fraction after pretreatment was addedand the post hydrolysis was run for 48 hours at 50° C. and pH 5.0 to5.3.

HPLC samples were taken daily to follow the conversion of cellulose andhem icellulose and analysed for glucose, xylose and arabinose using aDionex Ultimate 3000 HPLC system equipped with a Rezex Monosaccharidecolumn with quantification through use of external standard.

FIG. 7 shows hydrolysis data for conversion of hemicellulose withaddition of liquid fraction after 96 hours hydrolysis of solid fractionusing sugar cane bagasse pretreated to xylan number 12.3% and hydrolysedusing 0.3 ml Accellerase Trio™ (Genencor) per g glucan. Shown is atypical hydrolysis profile. C5 monomer recovery is expressed as apercent of theoretical yield from the material present in the hydrolysisreaction. Most of the hemicellulose within the solid fraction has beenconverted to monomeric sugars within the first 24 hours in hydrolysis ofthe solid fraction. Addition of liquid fraction after 96 hours increasesthe theoretical potential yield, which explains the drop in C5conversion observed just after liquid fraction is added. Within thefirst 24 hours most of the C5 from liquid fraction is converted tomonomers. Comparing the C5 conversion just before liquid fraction isadded with the end point of the hydrolysis, it is possible to calculatethe C5 conversion in the liquid fraction as 90% when using sugar canebagasse under these conditions.

Table 3 shows hydrolysis data for different biomasses pretreated underdifferent circumstances and hydrolysed using different dose levels of acommercially available cellulase preparation optimized forlignocellulosic biomass conversion, Accellerase Trio™ (Genencor). Allenzyme dose levels used were within the range suggested by themanufacturer. As shown, using single-stage autohydrolysis and enzymatichydrolysis with C5 bypass and post-hydrolysis, C5 monomer yields of 60%or greater can be achieved using manufacturers' recommended doses ofcommercially available cellulase preparations optimized forlignocellulosic biomass conversion while still achieving celluloseconversion of 60% or greater.

TABLE 3 Hydrolysis yields using very low severity single-stageautohydrolysis with C5 bypass and post-hydrolysis. WS SCB SCB CS CS EFBDry matter after soaking [wt %] 40% 39% 39% 40% 40% 39% Residence time[min] 12.0 12.0 12.0 12.0 12.0 12.0 Temperature [° C.] 183.0 182.7 182.7174.5 174.5 185.2 Pretreatment severity [logRo] 3.52 3.51 3.51 3.27 3.273.58 C5 recovery from pretreatment [%] 74% 87% 87% 88% 88% 84% Xylannumber 11.5% 12.3% 12.3% 15.6% 15.6% 15.5% Enzyme dosage [mL Ac. TRIO/gglucan] 0.2 0.3 0.3 0.3 0.2 0.4 % TS in fiber hydrolysis 22% 22% 22% 19%22% 22% Cellulose conversion after hydrolysis (96 h) 78% 64% 66% 68% 58%69% Hemicellulose conversion (C5 recovery) 80% 73% 73% 61% 61% 75% afterhydrolysis (96 h) % TS in second hydrolysis 18% 17% 17% 16% 18% 18Cellulose conversion after post hydrolysis 78% 65% 67% 67% 61% 72% (144h) Hemicellulose conversion (C5 90% 79% 78% 71% 68% 83% recovery)afterpost hydrolysis (144 h) Overall cellulose conversion 78% 65% 67% 67% 61%72% Overall C5 monomer yield 67% 69% 68% 63% 60% 70%

Example 7 Co-Fermentation to Ethanol of C5 and C6 Sugars in CombinedHydrolysate by Modified Yeast

As an example on the use of a hydrolysate produced from softlignocellulosic biomass (in this case wheat straw) prepared bysingle-stage autohydrolysis pretreatment to a xylan number >10%, FIG. 8shows data for a fermentation performed without detoxification or anyother process steps before fermentation with GMO yeast able to convertboth C5 and C6 sugars (strain V1 from TERRANOL™). The hydrolysate wasadjusted to pH 5.5 with KOH pellets before fermentation and supplementedwith 3 g/L urea. The fermentation was conducted as a batch fermentation.The initial cell concentration in the reactor was 0.75 g dw/L. Thefermentations were controlled at pH 5.5 using automatic addition of 10%NH3.

The temperature was kept at 30° C. and the stirring rate was 300 rpm. Asshown, glucose and xylose are readily consumed and ethanol readilyproduced, notwithstanding the presence of acetic acid, furfural andother compounds that would typically prove inhibitory at higher levelsof pretreatment severity.

Example 8 Experimental Determination of Activity Levels in CommercialCellulase Preparations

Commercial preparations of ACCELLERASE TRIO™ from GENENCOR™ and CELLICCTEC2™ and CELLIC CTEC3™ from NOVOZYMES™ were diluted so that proteinconcentrations were roughly equivalent in sample preparations tested.Equivalent volumes of diluted enzyme preparations were added and assaydeterminations made in duplicate or triplicate.

Assay of CBHI (exocellulase) activity was conducted in 50 mM NaOACbuffer at pH 5, 25° C., for 25 minutes. Activity was determined intriplicate by following continuous rate of 4-Methylumbelliferon release(Abs: 347 nm) from the model substrate4-methylumbelliferyl-β-cellobioside. Activity unit was 1 umole MeUmbequivalent/minute. Protein concentrations were 0.16, 0.14, 0.17mg/mIrespectively for CTEC3, ACTrio, and CTEC2 assays. Substrateconcentration was 0.5 mg/ml.

Assay of Endo-1,4-β-glucanase activity was conducted in 50 mM NaOACbuffer, pH 5; 50° C., for 60 minutes. Activity was determined intriplicate by following absorbance change associated with generation ofreducing ends from the model substrate Avicel PH-101. Activity unit was1 μmole glucose equivalent/min. Protein concentrations were 0.80, 0.67,0.79 mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrateconcentration was 80 mg/ml.

Assay of β-glucosidase activity was conducted in 50 mM NaOAC buffer, pH5; 50° C., for 20 minutes. Activity was determined in triplicate byfollowing absorbance change associated with release of glucose frommodel substrate cellobiose. Activity unit was 2 μmole glucose/min.Protein concentrations were 0.1, 0.12, 0.12 mg/ml respectively forCTEC3, ACTrio, and CTEC2 assays. Substrate concentration was 1.7 mg/ml.

Assay of Endo-1,4-β-xylanase activity was conducted in 50 mM NaOACbuffer, pH 5; 50° C., for 60 minutes. Activity was determined intriplicate by following absorbance change associated with generation ofreducing ends from the model substrate water extractable arabinoxylan.Activity unit was 1 μmole glucose equivalent/min. Protein concentrationswere 1.12, 0.97, 1.12 mg/ml respectively for CTEC3, ACTrio, and CTEC2assays. Substrate concentration was 10 mg/ml.

Assay of β-xylosidase activity was conducted in 50 mM NaOAC buffer, pH5; 50° C., for 60 minutes. Activity was determined in duplicate byfollowing release of xylose associated with hydrolysis of the modelsubstrate water extractable arabionxylan. Activity unit was 1 pmolexylose/min. Protein concentrations were 1.12, 0.97, 1.12 mg/m Irespectively for CTEC3, ACTrio, and CTEC2 assays. Substrateconcentration was 10 mg/ml.

Assay of β-L-arabinofuranosidase activity was conducted in 50 mM NaOACbuffer, pH 5; 50° C., for 60 minutes. Activity was determined intriplicate by following release of arabinoase associated with hydrolysisof the model substrate water extractable arabionxylan. Activity unit was1 μmole arabinose/min. Protein concentrations were 1.12, 0.97, 1.12mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrateconcentration was 10 mg/ml.

Assay of amyloglucosidase (AMG) activity was conducted in 50 mM NaOACbuffer, pH 5; 50° C., for 80 minutes. Activity was determined intriplicate by following absorbance change associated with glucoserelease from the model substrate soluble corn starch. Activity unit was1 pmole glucose/min. Protein concentrations were 1.12, 0.97, 1.12 mg/mlrespectively for CTEC3, ACTrio, and CTEC2 assays. Substrateconcentration was 10 mg/ml.

Assay of α-amylase activity was conducted in 50 mM NaOAC buffer, pH 5;50° C., for 60 minutes. Activity was determined in triplicate byfollowing absorbance change associated with generation of reducing endsfrom the model substrate soluble corn starch. Activity unit was 1 pmoleglucose equivalent/min. Protein concentrations were 1.12, 0.97, 1.12mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays. Substrateconcentration was 10 mg/ml.

Assay of acetyl xylan esterase activity was conducted in 100 mMSuccinate buffer, pH 5; 25° C., for 25 minutes. Activity was determinedin triplicate by following continuous rate of 4-Nitrophenyl release(Abs: 410 nm) from the model substrate 4 4-Nitrophenyl acetate. Activityunit was 1 μmole pNP equivalent/min. Protein concentrations were 0.48,0.42, 0.51mg/ml respectively for CTEC3, ACTrio, and CTEC2 assays.Substrate concentration was 10 mg/ml.

Results of the activity determinations are shown in Table 1.

The embodiments and examples are descriptive only and not intended tolimit the scope of the claims.

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1. A method of processing lignocellulosic biomass comprising: Providingsoft lignocellulosic biomass feedstock, Pretreating the feedstock at pHwithin the range 3.5 to 9.0 in a single-stage pressurized hydrothermalpretreatment to very low severity such that the pretreated biomass ischaracterized by having a xylan number of 10% or higher, Separating thepretreated biomass into an solid fraction and a liquid fraction,Hydrolysing the solid fraction with or without addition of supplementalwater content using enzymatic hydrolysis catalysed by an enzyme mixturecomprising endoglucanase, exoglucanase, B-glucosidase, endoxylanase,xylosidase and acetyl xylan esterase activities, and Subsequently mixingthe separated liquid fraction and the hydrolysed solid fraction, wherebyxylo-oligomers in the liquid fraction are degraded to xylose monomers bythe action of enzyme activities remaining within the hydrolysed solidfraction.